Site specifically incorporated initiator for growth of polymers from proteins

ABSTRACT

The present invention is directed towards a protein-polymer composition having a protein with a site-specifically incorporated unnatural amino acid initiator and a covalently attached polymer.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit of the filing date under 35 U.S.C.§119(a)-(d) of U.S. Provisional Patent Application No. 61/613,178, filedMar. 20, 2012, and is also a continuation-in-part of U.S. patentapplication Ser. No. 13/788,710, filed Mar. 7, 2013, which in turn is acontinuation of PCT International Application No. PCT/US2011/051043,filed Sep. 9, 2011, which in turn claims priority to U.S. ProvisionalApplication No. 61/381,757, filed Sep. 10, 2010.

INCORPORATION BY REFERENCE

Provisional application 61/381,757 filed on Sep. 10, 2010 is herebyincorporated by reference. PCT application PCT/US2011/51043 filed onSep. 9, 2011 is also hereby incorporated by reference.

GOVERNMENTAL INTEREST

Some of the work involved in the development described in the inventiondescribed in this patent application was partially funded by theNational Science Foundation grant DMR-09-69301.

FIELD OF THE INVENTION

The present invention is directed towards a protein-polymer compositionhaving a site-specifically incorporated unnatural amino acid initiatorand a covalently attached polymer, and a general method for producingthe composition through controlled radical polymerization.

BACKGROUND

Protein-polymer hybrids have revolutionized the treatment of disease[Chemical Reviews, 2009, 109, 5402-5436; Nat Rev Drug Discov, 2003, 2,347-360] and biocatalytic processes. [J. Am. Chem. Soc., 2006, 128,11008-11009]. Protein-polymer hybrids typically comprise linear orbranched polymers “grafted to” or “grafted from” accessable sites withinthe desired protein. These protein-polymer hybrids have already shown animpressive range of altered or improved properties. From a therapeuticperspective, the advantages of protein-polymer hybrids over nativeproteins include increased in vivo stability, minimized immunerecognition due to steric effects, enhanced in vivo circulation, andimproved therapeutic effects. Protein-polymer hybrids have also shown anincreased solubility in non-aqueous media, which have expanded theutility of enzymatic biocatalytic processes into the realm of organicsynthesis. [Biomacromolecules, 2009, 10, 1612-1618; BiotechnologyProgress, 1994, 10, 398-402]

Recently, the concept of protein-polymer nanogel hybrids has beenintroduced in order to overcome some of the long-term stability issuesassociated with protein-polymer hybrids. [J. Am. Chem. Soc. 2006, 128,11008-11009; J. Phys. Chem. B, 2008, 112, 14319-14324; J. Biotechnology2007, 128, 597-605.] Some of these issues include organic solventsolubility and deactivation of traditional protein-polymer hybrids underharsh conditions. Both of these characteristics are extremely importantfor expanding the catalytic potential of enzymatic systems.Encapsulation of proteins into nanogel matrices have demonstratedsuperior temperature and organic solvent stability for several systems,such as carbonic anhydrase, lipase, and horseradish peroxidase amongothers. [Biomacromolecules, 2007, 8, 560-565 and 2009, 10, 1612-1618; J.Am. Chem. Soc. 2006, 128, 11008-11009; Angew. Chem., Int. Ed, 2008, 47,6263-6266]

Traditionally, protein-polymer hybrids are synthesized in a two-stepprocess. The proteins are first functionalized withN-hydroxysuccinimide-acrylate and then copolymerized with an acrylamideand a crosslinker using REDOX initiated free radical polymerization.However, this process produces uncontrolled, non-specific acrylatefunctionalization of the protein, and often leads to batch-to-batchvariability of protein activity. This variability often originates fromnon-specific modification of lysine residues by the acrylate chemistry,resulting in deactivation of active sites and protein denaturing. [NatRev Drug Discov, 2003, 2, 214-221] Additionally, the polymers accessiblethrough REDOX initiated free radical polymerizations are limited by anumber of factors, including monomer selection, particle size, proteinloading, and potential for controlled release properties.

More recently, protein-polymer hybrids have been prepared usingcontrolled radical polymerization techniques (see Wang et al., Am. Chem.Soc. 1995, 117, 5614; Matyjaszewski & Xia, Chem. Rev. 2001, 101, 2921(“Xie”); Matyjaszewski &Tsarevsky, Nature Chem. 2009, 1, 276) whichallow unprecedented control over polymer dimensions (molecular weight),uniformity (polydispersity), topology (geometry), composition andchemical functionality. [Matyjaszewski, K., Ed, Controlled RadicalPolymerization; ACS: Washington, D.C., 1998; ACS Symposium Series 685.Matyjaszewski, K., Ed.; Controlled/living Radical Polymerization.Progress in ATRP, NMP, and RAFT; ACS: Washington, D.C., 2000; ACSSymposium Series 768; Matyjaszewski, K., Davis, T. P., Eds. Handbook ofRadical Polymerization; Wiley: Hoboken, 2002; Qiu, J.; Charleux, B.;Matyjaszewski, K. Prog. Polym. Sci. 2001, 26, 2083; Davis, K. A.;Matyjaszewski, K. Adv. Polym. Sci. 2002, 159, 1.]

While controlled radical polymerization techniques permit greatercontrol over the polymer's composition, there is still a need formethods to attach those polymers to site-specific locations on aprotein. Thus far, methods for site-specific incorporation ofpolymerization initiators into proteins have been limited to theN-terminal position or specific natural amino-acid directed linkages.Both of these suffer from challenging purification of intermediatesand/or the inability to efficiently control the number or location ofpotential polymer connections, both of which can compromise thestructural integrity of the modified protein.

While the many experiments conducted using in situ functionalizednatural amino acids on proteins have illustrated the potential immenseimpact of well-defined protein-polymer hybrids, their application islimited by technical shortcomings, and there is a need to developprotein polymer hybrids where a desired polymer can be attached at asite-specific location on the protein. [See Broyer et al., J. Am. Chem.Soc. 2008, 130, 1041]

SUMMARY

In view of the above-mentioned need, a protein-polymer composition isprovided. The protein-polymer composition includes a protein with asite-specifically incorporated unnatural amino acid with a covalentlyattached polymer.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the invention will be understood by reference to thefollowing figures, wherein:

FIG. 1: Shows the emission spectra for GFP-wt and GFP-1-p(OEO300MA) ˜510nm

FIG. 2: Formation of a catalase protein-nanogel particle in an inverseminiemulsion (Synthetic Scheme 1).

FIG. 3: A) FIG. 3.1 Dynamic light scattering of GFP-1 peak size ˜240 nm.B) FIG. 3.2 confocal microscopy of GFP-1.

FIG. 4: Fast Protein Liquid Chromatography (“FPLC”) of GFP-1 reaction,GFP-1 (0 min), GFP-1-p(OEO300MA) (180 min).

FIG. 5: Lower Critical Solution Temperature (“LCST”) behavior of GFP-1-p(OEO300MA)

FIG. 6: Shows IMAGE 1, a Catalase-NG, before and after H2O2 addition,bioengineered to possess 4 ATRP initiating groups.

FIG. 7: Shows IMAGE 2, an AFM of a sample of p(GFP-2-PEO_(lk)dialkyne)cast at an angle on the surface of mica where a long fiber-like materialhad formed.

FIG. 8: Shows “Grafting From” GFP-1, where an exemplary unnatural aminoacid, 4,(2′-bromoisobutylamido)-phenylalanine, was produced by replacingGFP-wt's Asp-134 (Synthetic Scheme 2).

FIG. 9: Shows expression levels of GTP-wt from pBad-GFP-His6 (SyntheticScheme 3).

DETAILED DESCRIPTION OF THE EMBODIMENT(S)

One aspect of the invention is a protein-polymer composition having aprotein with a site-specifically incorporated unnatural amino acid witha covalently attached polymer.

The general method of preparing a protein with a site-specificallyincorporated unnatural amino acid is disclosed by Mehl et al.,PCT/US2011/57043, and is incorporated herein by reference.

A “protein” (or portion thereof) is understood to include nativeproteins, as well as proteins that have one or more site-specificallyincorporated unnatural amino acids further comprising an initiator for aCRP. No attempt is made to identify the hundreds of thousands of knownproteins, any of which may be modified to include one or more unnaturalamino acid initiators, e.g., by tailoring any available mutation methodsto include one or more appropriate selector codon in a relevanttranslation system. Common sequence repositories for known proteinsinclude GenBank, EMBL, DDBJ, and the NCBI, among others. Typically, theproteins are, e.g., at least 60%, at least 70%, at least 75%, at least80%, at least 90%, at least 95%, or at least 99% or more identical toany available protein (e.g., a therapeutic protein, a diagnosticprotein, an industrial enzyme, or portion thereof, and the like), andthey can comprise one or more unnatural amino acid initiators.Essentially any protein of interest can be modified to include aninitiator comprising an unnatural amino acid initiator.

Proteins are also understood to include enzymes (e.g., therapeutic,diagnostic, or industrial enzymes), or portions thereof with at leastone or more unnatural amino acid initiators are also provided by theinvention. Examples of enzymes include, but are not limited to, e.g.,amidases, amino acid racemases, acylases, dehalogenases, dioxygenases,diarylpropane peroxidases, epimerases, epoxide hydrolases, esterases,isomerases, kinases, glucose isomerases, glycosidases, glycosyltransferases, haloperoxidases, monooxygenases (e.g., p450s), lipases,lignin peroxidases, nitrile hydratases, nitrilases, proteases,phosphatases, subtilisins, transaminases, and nucleases.

The term “selector codon” refers to a codon recognized by the O-tRNA inthe translation process and not typically recognized by an endogenoustRNA. The O-tRNA anticodon loop recognizes the selector codon on themRNA and incorporates its amino acid, e.g., an initiator amino acid, atthis site in the polypeptide. Selector codons can include, e.g.,nonsense codons, such as stop codons (e.g., amber, ochre, and opalcodons), four or more base codons, rare codons, codons derived fromnatural or unnatural base pairs, or the like.

The term “translation system” refers to the components that incorporatean amino acid into a growing polypeptide chain (protein). Components ofa translation system can include, e.g., ribosomes, tRNAs, synthetases,mRNA, and the like. Typical translation systems include cells, such asbacterial cells (e.g., Escherichia coli), archeaebacterial cells,eukaryotic cells (e.g., yeast cells, mammalian cells, plant cells,insect cells), or the like. Alternatively, the translation systemcomprises an in vitro translation system, e.g., a translation extractincluding a cellular extract. The O-tRNA or the O-RSs of the inventioncan be added to or be part of an in vitro or in vivo translation system,e.g., in an eukaryotic cell, e.g., a bacterium (such as E. coli), or ina eukaryotic cell, e.g., a yeast cell, a mammalian cell, a plant cell,an algae cell, a fungus cell, an insect cell, or the like. Thetranslation system can also be a cell-free system, e.g., any of avariety of commercially available in vitro transcription/translationsystems in combination with an O-tRNA/O-RS pair and an initiator aminoacid as described herein.

The translation system may optionally include multiple O-tRNA/O-RSpairs, which allow incorporation of more than one unnatural amino acid,e.g., an initiator amino acid and another unnatural amino acid. Forexample, the cell can further include an additional differentO-tRNA/O-RS pair and a second unnatural amino acid, where thisadditional O-tRNA recognizes a second selector codon and this additionalO-RS preferentially aminoacylates the O-tRNA with the second unnaturalamino acid. For example, a cell that includes an O-tRNA/O-RS pair (wherethe O-tRNA recognizes, e.g., an amber selector codon) can furthercomprise a second orthogonal pair, where the second O-tRNA recognizes adifferent selector codon (e.g., an opal codon, four-base codon, or thelike). Desirably, the different orthogonal pairs are derived fromdifferent sources, which can facilitate recognition of differentselector codons.

An “unnatural amino acid” is, in this case a molecule containing aprimary amine functionality and carboxylic acid functionality that canbe incorporated into a protein primary sequence with a transferable atomor group that is completely incorporated into the final product.

In an embodiment, the unnatural amino acid is site-specificallyincorporated into the protein one to five times. In another exemplaryembodiment, the unnatural amino acid is site-specifically incorporatedinto a protein one to three times. In yet another exemplary embodiment,the unnatural amino acid is site-specifically incorporated into aprotein one to two times. Exemplary examples include GFP-1, discussedbelow, wherein a single unnatural amino acid with a covalently attachedpolymer was incorporated without loss of fluorescence. FIG. 1. Inanother exemplary example, incorporation of Catalase-NG with fourincorporated initiation sites into a nanogel was accomplished whileretaining the ability of Catalase to reduce hydrogen peroxide. FIG. 2and Image 1, FIG. 6.

As used herein, the term “nanogel” refers to a polymer networkdispersion capable of absorbing a fluid and retaining at least a portionof the fluid to form a swollen polymer particle. A nanogel can have manysizes, and these sizes are indicative of the nanogel in solvent swollenform.

In an exemplary embodiment, the site specifically incorporated unnaturalamino acid is an initiator for a controlled radical polymerizationreaction (“CRP”). CRP reactions include, but are not limited to, atomtransfer radical polymerization (“ATRP”), nitroxide mediatedpolymerization (“NMP”), and reversible addition fragmentation transfer(“RAFT”) systems. CRP reactions allow unprecedented control over polymerdimensions (molecular weight), uniformity (polydispersity), topology(geometry), composition and functionality. [Matyjaszewski, K., Davis, T.P., Eds. Handbook of Radical Polymerization; Wiley; Hoboken, 2002; Qiu,J.; Charleux, B.; Matyjaszewski, K. Prog. Polym. Sci, 2001, 26, 2083;Davis, K. A.; Matyjaszewski, K. Adv. Polym. Sci, 2002, 159, 1.]

Matyjaszewski and coworkers disclosed the fundamental four componentAtom Transfer Radical Polymerization (ATRP) process comprising theaddition, or in situ formation, of an initiator, in this case a moleculewith a transferable atom or group that is completely incorporated intothe final product, a transition metal and a ligand that form, apartially soluble transition metal complex that participates in areversible redox reaction with the added initiator or a dormant polymerto form the active species to copolymerize radically polymerizablemonomers, and a number of improvements to the basic ATRP process, in anumber of patents and patent applications: U.S. Pat. Nos. 5,763,546;5,807,937; 5,789,487; 5,945,491; 6,111,022; 6,121,371; 6,124,411;6,162,882; 6,624,262; 6,407,187; 6,512,060; 6,538,091; 6,541,580;6,624,262; 6,627,314; 6,759,491; 6,790,919; 6,887,962; 7,019,082;7,049,373; 7,064,166; 7,125,938; 7,157,530; 7,332,550 and U.S. patentapplication Ser. Nos. 09/534,827; PCT/US04/09905; PCT/US05/007264;PCT/US05/007265; PCT/US06/33152; PCT/US2006/048656 and PCT/US08/64710,all of which are herein incorporated by reference to provide bothbackground and definitions for the terms used herein. Papers includeWang et al., Am. Chem. Soc. 1995, 117, 5614; Matyjaszewski & Xia, Chem.Rev. 2001, 101, 2921; Matyjaszewski & Tsarevsky, Nature Chem. 2009, 1,276.

In an exemplary embodiment, the unnatural amino acid is an initiator forATRP, therefore allowing for monomers and cross-linkers to beincorporated in a predictable, controlled, and programmed manner toyield polymer chains of essentially equal length, as defined by theratio of consumed monomer to the added initiator. Moreover, thefunctionality present on the introduced initiator can be preserved,including both the α- and ω-chain end functionality on the formedpolymer segment. The polymers synthesized using ATRP also may allow manyfunctional groups, such as hydroxyl, amino, amido, esters, carboxylicacid, to be incorporated into a copolymer for use in post-polymerizationmodifications, including covalent linking of biomolecules for drugdelivery. As disclosed below, this enables formation of protein-polymerhybrids between synthetic polymers and biomolecules, and providesdelivery systems with customizable and tunable polymer structures formany applications, including but not limited to precise targeteddelivery of biologically active molecules.

An “initiator” is understood to mean a chemical species with atransferable atom that is capable of interacting with a transition metaland a ligand to form a partially soluble transition metal complex thatparticipates in a reversible redox reaction with the added initiator ora dormant polymer to form the active species to copolymerize radicallypolymerizable monomers.

An exemplary embodiment, the unnatural amino acid is represented byformula 2:

wherein R1 and R2 are independently H, C1-C8 alkyl, cycloalkyl,heterocycloalkyl, aryl, or heteroaryl; X is F, Cl, Br, I, N₃,alkoxyamine, or a thiocarbonyl thio moiety; A is O, S, or NR, wherein Ris H, C1-C8 alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl;and as is 0, 1, 2, or 3; or a salt thereof.

In another exemplary embodiment, the unnatural amino acid is representedby formula 1:

wherein X is F, Cl, Br, I, or —N₃, or a salt thereof. In anotherembodiment, the unnatural amino acid is represented by formula 1,wherein X is Br or Cl, or a salt thereof. In yet another embodiment, theunnatural amino acid is represented by formula 1, wherein X is —N₃ or asalt thereof.

In another embodiment, the protein-polymer composition has a polymerwith repeating units from a monomer class including methacrylates,acrylates, acrylamides, styrenics, or acrylamide-styrenics, orcombinations thereof.

Another aspect of the invention is that the polymer in theprotein-polymer composition can include a copolymer that has repeatingunits from a monomer class including methacrylates, acrylates,acrylamides, styrenics, or acrylamide-styrenics, or combinationsthereof.

In yet another embodiment, the polymer may be copolymerized withdifunctional monomers.

In yet another embodiment, when the polymer is copolymerized withdifunctional monomers, and the protein is incorporated into ahyperbranched structure or a nanogel.

In yet another embodiment, the polymer employed in the protein-polymercomposition can be degradable. In one non-limiting exemplary embodiment,a single linkage point between the protein and the polymer network canallow the protein to be efficiently released from the conjugated polymerby cleaving a degradable link, e.g. disulfide or acetal, as each proteinis attached to the network through only one link/chain, thereby makingthese protein-polymer conjugates better suited for controlled releaseapplications. In another non-limiting exemplary embodiment, multiplelinkage points between the protein and the polymer network can provideadditional control over efficiently releasing the conjugated polymer bycleaving the degradable linkages.

In an exemplary embodiment, an injectable protein-nanogel is preparedwhen copolymerization is conducted in an emulsion. (Scheme 1, FIG. 2)

In another embodiment, a protein-polymer nanogel hybrid is providedhaving a protein linked to one primary polymer chain at a precise regionof the protein, thereby providing greater scaffold structural integritywhilst still forming well defined particles due to the emulsion processemployed for the synthesis of the nanogel.

In additional embodiments, a targeted protein-polymer nanogel system isprepared using programmable behaviors of thermo-responsive or a pHsensitive composite structure. For example, as an exemplary embodiment,hydroxyethylmethacrylate (HEMA) can be chosen as a monomer for thepolymerization process to provide available functional groups forfurther post-polymerization reactions and give a route for hydrogelsynthesis.

In another exemplary embodiment, the polymer can be a cross-linkedpolymer. In such an embodiment, a degradable crosslinker can be used inthe synthesis, which results in the preparation of nanogels that providefor a controlled release of proteins and bio-active molecules. Exemplaryembodiments include, but are not limited to, deliveries ofpolynucleotides (e.g. oligonucleotides) and/or other therapeutic agentsfrom the protein-polymer hybrids. This can be important for drugdelivery applications through extended t½ life circulation of proteintherapeutics.

In yet another embodiment, protein-polymer hybrids can incorporatedifferent proteins at have synergistic activity, and can providemulti-protein nanogels with distinct protein domains within the nanogel.These systems have greater stability to enzymatic degradation whileproviding for controlled release of tethered bio-active agents ifdegradable cross-linkers are used, and also can have increased stabilityin organic solvents as compared to isolated wild-type proteins orwild-type proteins simply entrapped in a nanogel. Scheme 1 and FIG. 2show an exemplary embodiment having a protein incorporated into ananogel. These systems are proposed to have greater stability toenzymatic degradation, and increased potential for controlled release ifdegradable cross linkers are employed.

Another aspect of the invention is a method for preparing aprotein-polymer composition having a protein with a site-specificallyincorporated unnatural amino acid covalently attached to a polymer. Themethod comprises the steps of:

Providing a first protein containing a site specifically incorporatedunnatural amino acid initiator, a polymerization catalyst precursor, andan organic solvent to an aqueous solution to form an emulsion;

providing a first radically polymerizable monomer to the emulsion; and

providing a catalyst precursor reducing agent is added to the emulsion.

The process is exemplified by using a protein with a site-specificallyincorporated unnatural amino acid with a CRP initiator functionality.Exemplary embodiments include, but are not limited to, CRP initiatorsfor ATRP, NMP, or RAFT. These initiators can be introduced into nearlyany protein thereby providing the ability to advance the field ofprotein polymer hybrids from non-functional proteins, e.g. bovine serumalbumin, towards enzymes or therapeutically relevant systems. Therefore,one can assay the efficacy of the system and properly study the effectsof polymer placement using commercially available enzyme assays.

In an embodiment, the method is exemplified by an unnatural amino acidinitiator of formula 2, wherein R1 and R2 are independently H, C1-C8alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; X is F, Cl,Br, I, N₃, alkoxyamine, or a thiocarbonyl thio moiety; A is O, S, or NR,wherein R is H, C1-C8 alkyl, cycloalkyl, heterocycloalkyl, aryl, orheteroaryl; and n is 0, 1, 2, or 3; or a salt thereof.

In another embodiment of the method, the unnatural amino acid initiatoris of formula 1, wherein X is a F, Cl, Br, I, or —N₃, or a salt thereof.In yet another embodiment of the method, the unnatural amino acidinitiator is of formula 1, wherein X is —N₃, or a salt thereof. Inanother embodiment of the method, the unnatural amino acid initiator isof formula 1, wherein X is Br or Cl, or a salt thereof.

In one embodiment, a coinitiator is added to the emulsion. In anadditional embodiment, polyethyleneglycolisobutyryl bromide is acoinitiator and is added to the emulsion. In another embodiment, asecond protein containing a site-specifically incorporated unnaturalamino acid initiator is added to the emulsion.

In another embodiment, the polymerization catalyst precursor is atransition metal and a transition metal ligand species, that form apartially soluble transition metal complex that participates in areversible redox reaction with the added initiator to form an activespecies suitable for polymerization of a radically polymerizablemonomer. In an exemplary embodiment, the polymerization catalystprecursor comprises a copper halide and a transition metal ligandspecies. In another exemplary embodiment, the copper halide is CuBr₂ orCuCl₂.

Exemplary examples of radically polymerizable monomers include, but arenot limited to methacrylates, acrylates, acrylamides, styrenics, oracrylamide-styrenics, or combinations thereof. In one embodiment, themethod further comprises adding a radically polymerizable copolymer tothe emulsion. The radically polymerizable copolymer can have repeatingmonomers. In an embodiment, the copolymer monomers can includemethacrylates, acrylates, acrylamides, styrenics, oracrylamide-styrenics, or combinations thereof. In another embodiment,the polymer is copolymerized with difunctional monomers. In anotherembodiment, the first radically polymerizable monomer is added to theemulsion continuously or in stages during the polymerization process. Inanother embodiment, the radically polymerizable copolymer is added tothe emulsion continuously or in stages during the polymerizationprocess.

A catalyst precursor reducing agent for CRP reactions may be anyreducing agent capable of reducing the transition metal catalyst from ahigher oxidation state to a lower oxidation state, such as, but notlimited to, ascorbic acid or salts thereof; tin octonate, reducingsugars such as fructose, antioxidants, those used in food preservativessuch as flavonoids, beta carotene, α-tocopherol, propyl or octyl gallate(triphenol) BHA or BHT, or other food preservatives such as nitrites,propionic acids, sorbates, or sulfites. In another embodiment, thecatalyst precursor reducing agent is ascorbic acid.

In another embodiment, the method further comprises the step of adding across-linking reagent to the emulsion. Exemplary example of across-linking reagent include, but are not limited to methacrylates,acrylates, acrylamides, styrenics, or acrylamide-styrenics, orcombinations thereof. In another embodiment, when a cross-linkingreagent is added to the emulsion, a coinitiator is further added to theemulsion.

To demonstrate the utility of a protein-polymer hybrid having asite-specifically incorporated unnatural amino acid covalently attachedto a polymer, a green fluorescent protein (“GFP”) with the functionalinitiating site specifically incorporated on sample GFP-1's surface wasproduced as a non-limiting exemplary protein. See scheme 2, FIG. 8.Using an exemplary unnatural amino acid,4-(2′-bromoisobutylamido)-phenylalanine, GFP-1 was produced by replacingGFP-wt's Asp-134 through a variation of the procedure disclosed in U.S.Pat. No. 7,776,535, which is incorporated by reference. Specifically,the exemplary unnatural amino acid was incorporated into a methanococcusjannaschii (Mj) tyrosyltRNA synthetase (RS)/tRNACUA pair to geneticallyencode this initiator in response to an amber codon. Grafting from theincorporated 4-(2′-bromoisobutylamido)-phenylalanine initiator understandard ATRP conditions with the monomer oligo(ethylene oxide)monomethyl ether methacrylate, did not affect the green fluorescentproperties of the GFP protein, allowing the fluorescent properties to bea measure of the influence of the conditions employed for the CRP on thestructure of the selected protein. FIG. 1 shows that the fluorescentproperties were not affected. Polymers grown from random sites on anunmodified sample of GFP-wt resulted in a lack of fluorescent propertiesby the GFP-polymer hybrid.

The exemplary GFP-1 proteins used in this work were attached to apolymer by a single covalent linkage to the site-specificallyincorporated initiator, and the GFP-1 was incorporated into theresulting nanogel. The nanogel incorporated GFP-1 protein was notcompromised by either the covalent unnatural amino acid-polymer linkageor subsequent incorporation into a nanogel, which was confirmed by thefact that the nanogels retained GFP's intrinsic light emittingproperties, FIG. 3.2.

Other embodiments of the invention include the production of functionalprotein-polymer hybrid materials such as enzymes and assaying theiractivity under synthetically relevant conditions. This ability toprepare functional proteins with site selected functionality is furtherexemplified by preparation of a Catalase that was bio-engineered topossess 4 ATRP initiating groups, sample Cat-1 FIG. 2. Catalase is anenzyme that converts H₂O₂ into oxygen and water and it was confirmedthat the enzymatic reaction was not modified after the Catalase hadundergone polymer modification, Image 1, FIG. 6.

One skilled in the art should appreciate that the above steps are merelyexemplary and used to enable one skilled in the art to prepareprotein-polymer compositions containing a protein with site-specificallyincorporated unnatural amino acid covalently attached to a polymer,Additionally, those skilled in the art will recognize, or be able toascertain using no more than routine experimentation, numerousequivalents to the specific procedures, embodiments, claims, andexamples described herein. Such equivalents were considered to be withinthe scope of this invention and covered by the claims appended hereto.

EXAMPLES Example 1: Synthesis of Protein-Polymer Conjugates Via“Grafting from” an Incorporated Functionality

The procedure used to incorporate a functional group that can act as aninitiator for an ATRP into a protein is schematically represented inScheme 3, FIG. 9. The first formed ATRP initiator4-(2′-bromoisobutyramido) phenylalanine (1) need not be asymmetric,since the MjRS utilizes only the L form. (B, FIG. 9.2).

This was accomplished using an E. coli tyrosyl tRNA/tRNA-synthesize pairvector. By using genetic engineering, specific placement of theinitiating amino acid was selected to be expressed at the 134 positionof GFP, the resulting modified protein being GFP-1, protecting theprotein's active sites and structurally weak regions.

N-Boc-4-(2′-bromoisobutyramido)-phenylalanine

Commercially obtained N-Boc-4-aminophenylalanine (3.62 g, 0.01447 mol)was dissolved in 50 mL of dry THF, 2-bromoisobutyryl bromide (1.757 mL,0.01422 mol) was added dropwise over 30-60 seconds with vigorousstirring. The reaction was complete after 10 min (monitored by TLC).After approximately 20 min. the entire reaction mixture (including newlyformed precipitate) was transferred to a separatory funnel with CHCl₃,and approximately 100 mL of H₂O. The reaction mixture was extracted withCHCl₃ (3×50 mL). The organic phase was washed with distilled water (2×50mL) and brine (50 mL). The organic phase was dried with MgSO₄ andevaporated in vacuo to obtain the crude product (4.55 g). The crudesolid product was recrystallized in 20-30 acetonitrile three times topurify the product. After three recrystallizations, the desired productwas obtained in 65% yield (3.63 g). ¹H NMR (500 MHz, DMSO) confirmed thestructure.

4-(2′-bromoisobutyramido)phenylalanine

N-Boc-4-(2′-bromoisobutyramido)-phenylalanine (4.8 g, 0.0112 mol) wasdissolved in 50 mL ethyl acetate under argon and dry 4 M HCl in dioxane(50 mL) was subsequently added to the solution while stirring at roomtemperature overnight. The reaction mixture was then evaporated underreduced pressure to a final volume of 5-10 mL. Pentane was then added tothe solution, and the precipitate was filtered using an M type filtercrucible and dried under reduced pressure. The product was present asthe HCl salt in 97% yield (3.93 g). ¹H NMR (500 MHz, DMSO) confirmed thestructure.

Selection of an Aminoacyl-tRNA Synthetase Specific for4-(2′-bromoisobutyramido)phenylalanine

The library of aminoacyl-tRNA synthetases was encoded on a kanamycin(Kn) resistant plasmid (pBK, 3000 bp) under control of the constitutiveEscherichia coli GlnRS promoter and terminator. The aminoacyl synthetaselibrary (3D-Lib) was randomized as follows: Leu65, His70, Gln155, andIle159 were randomized to all 20 natural amino acids; Tyr32 wasrandomized to 15 natural amino acids (less Trp, Phe, Tyr, Cys, and Ile);Asp158 was restricted to Gly, Ser, or Val; Leu162 was restricted to Lys,Ser, Leu, His, and Glu; and Phe108 and Gln109 were restricted to thepairs Tip-Met, Ala-Asp, Ser-Lys, Arg-Glu, Arg-Pro, Ser-His, or Phe-Gln.The library plasmid, pBK-3D-Lib, was moved between cells containing apositive selection plasmid (pCG) and cells containing a negativeselection plasmid (pNEG).

The positive selection plasmid, pCG (10000 bp), encodes a mutantMethanococcus jannaschii (Mj) tyrosyl-tRNACUA, an amber codon-disruptedchloramphenicol acetyltransferase, an amber codon-disrupted T7 RNApolymerase that drives the production of green fluorescent protein, andthe tetracycline (Tet) resistance marker. The negative selectionplasmid, pNEG (7000 hp), encodes the mutant tyrosyl-tRNACUA, an ambercodon-disrupted barnase gene under control of an arabinose promoter andrrnC terminator, and the ampicillin (Amp) resistance marker. pCGelectrocompetent cells and pNEG electrocompetent cells were made fromDH10B cells carrying the respective plasmids and stored in 100 μLaliquots at −80° C. for future rounds of selection.

The synthetase library in pBK-3D-Lib was transformed by electroporationinto DH10B cells containing the positive selection plasmid, pCG. Theresulting pCG/pBK-3D-Lib-containing cells were amplified in 1 L of 2×YTwith 50 μg/mL Kn and 25 μg/mL Tet with shaking at 37° C. The cells weregrown to saturation, then pelleted at 5525 ref, resuspended in 30 mL of2×YT and 7.5 mL of 80% glycerol, and stored at −80° C. in 1 mL aliquotsfor use in the first round of selections.

For the first positive selection, 2 mL of pCG/pBK-3D-Lib cells werethawed on ice before addition to 1.2 L of room temperature 2×YT mediacontaining 50 μg/mL Kn and 25 μg/mL Tet. After incubation (11 h, 250rpm, 37° C.), a 200 μL aliquot of these cells was plated on eleven 15 cmGMML-agar plates containing 50 μg/mL Kn, 25 μg/mL Tet, and 60 μg/mL,chloramphenicol (Cm). The positive selection agar medium also contained1 mM 1. After spreading, the surface of the plates was allowed to drycompletely before incubation (37° C., 15 h). To harvest the survivinglibrary members from the plates, 10 mL of 2×YT (50 μg/mL Kn, 25 μg/mLTet) was added to each plate. Colonies were scraped from the plate usinga glass spreader. The resulting solution was incubated with shaking (60min, 37° C.) to wash cells free of agar. The cells were then pelleted,and plasmid DNA was extracted. For the first positive selection a Qiagenmidiprep kit was used to purify the plasmid DNA. For all other plasmidpurification steps a Qiagen miniprep kit was used to purify the plasmidDNA. The smaller pBK-3D-Lib plasmid was separated from the larger pCGplasmid by agarose gel electrophoresis and extracted from the gel usingthe Qiagen gel extraction kit.

The purified pBK-3D-Lib was then transformed into pNEG-containing DH10Bcells. A 100 μL sample of pNEG electrocompetent cells was transformedwith 50 ng of purified pBK-3D-Lib DNA. Cells were rescued in 1 mL of SOCfor 1 h (37° C., 250 rpm) and the entire 1 mL of rescue solution wasplated on three 15 cm LB plates containing 100 μg/mL Amp, 50 μg/mL Kn,and 0.2% L-arabinose. Cells were collected from plates and pBK-3D-Libplasmid DNA was isolated in the same manner as described above forpositive selections.

For the second round of positive selection, 50 ng of purified libraryDNA was transformed into 100 μL of pCG competent cells. Thetransformants were rescued for 1.5 h in 1 mL of SOC (37° C., 250 rpm). A50 sample of these cells was plated on three plates prepared asdescribed in the first positive selection on LB agar plates.

For the second negative selection, one plate was spread with 250 μL ofrescued cells, and two plates were spread with 50 μL of rescued cellsand then incubated (12-16 h, 37° C.). For this round, the cells wereplated on LB agar containing 100 μg/mL Amp, 50 μg/mL Kn, and 0.04%L-arabinose.

In order to evaluate the success of the selections based on variation insynthetase efficacy (as opposed to traditional survival/death results),the synthetases resulting from the selection rounds were tested with thepALS plasmid. This plasmid contains the sfGFP reporter with a TAG codonat residue 150 as well as tyrosyl-tRNACUA. When a pBK plasmid with afunctional synthetase is transformed with the pALS plasmid and the cellsare grown in the presence of the appropriate amino acid on autoinductionagar, sfGFP is expressed and the colonies are visibly green.

One microliter of each library resulting from the second positive andthe second negative rounds of selection was transformed with 60 μL, ofpALS-containing DH10B cells. The cells were rescued for 1 hr in 1 mL ofSOC (37° C., 250 rpm). A 250 μL and 50 μL of cells from each librarywere plated on autoinducing minimal media with 25 μg/mL Kn, 25 μg/mLTet, and 1 mM 1. Plates were grown at 37° C. for 24 hours and then grownon the bench top, at room temperature, for an additional 24 hours.

Autoinducing agar plates were prepared by combining the reagents inTable 1A with an autoclaved solution of 40 g of agarose in 400 mL water.Sterile water was added to a final volume of 500 mL. Antibiotics wereadded to a final concentration of 25 μg/mL Tet and 25 μg/mL Kan. Nineplates were poured with 1 mM 1, and nine plates were maintained ascontrols without UAA.

A total of 92 visually green colonies were selected from the two 1 mM 1plates and used to inoculate a 96-well plate containing 0.5 mL per wellnon-inducing minimal media (Table 1B, with sterile water added to afinal volume of 500 mL) with 25 μg/mL Kn, 25 μg/mL Tet. After 24 hoursof growth (37° C., 250 rpm), 5 μL of these non-inducing samples wereused to inoculate 96-well plates with 0.5 mL autoinduction media (Table1C, with sterile water added to a final volume of 500 mL) containing 25μg/mL Kn, 25 μg/mL Tet with and without 1 mM 1. Fluorescencemeasurements of the cultures were collected 40 hours after inoculationusing a HORIBA Jobin Yvon FluoroMax®-4. The emission from 500 to 520 nm(1 nm bandwidth as summed with excitation at 488 nm (1 nm bandwidth).Samples were prepared by diluting suspended cells directly from culture100-fold with phosphate buffer saline (PBS).

TABLE 1 Components for autoinducing and non-inducing mediums, for finalvolume of 500 mL. A) Auto- B) Non- C) Auto- induction inducing inducingmedium medium plates 5% aspartate, pH 7.5 25 mL 25 mL 25 mL 10% glycerol25 mL 25 mL 25× 18 amino acid mix 20 mL 20 mL 20 mL 50× M 10 mL 10 mL 10mL leucine (4 mg/mL), pH 7.5 5 mL 5 mL 5 mL 20% arabinose 1.25 mL — 1.25mL 1M MgSO₄ 1 mL 1 mL 1 mL 40% glucose 625 μL 6.25 mL 125 μL Tracemetals 100 μL 100 μL 100 μL

Fluorescence measurements of 92 synthetases with GFP clones wereconducted. Expressions of 500 μL were grown for 40 hours before dilutionof suspended cells directly from culture 100-fold with phosphate buffersaline (PBS). Fluorescence measurements were collected using a HORIBAJobin Yvon FluoroMax®-4. The emission from 500 to 520 nm (1 nmbandwidth) was summed with excitation at 488 nm (1 nm bandwidth).

Fluorescence Analysis of Highest-Fluorescing Clones

Non-inducing cultures (3 mL) with 25 μg/mL Kn and 25 μg/mL Tet weregrown to saturation (37° C. with shaking at 250 rpm) from the 20highest-fluorescing colonies. Autoinduction cultures (3 mL) with 25μg/mL Kn and 25 μg/mL Tet were inoculated with 30 μL of non-inducingcultures and grown with and without 1 mM 1 at 37° C. with shaking at 250rpm. After approximately 40 hours, fluorescence was assessed. The topeight performing clones were sequence revealing five unique members. Thetop performing clone (G2) was moved from the pBK-G2 plasmid to the pDuleplasmid (PDule-BIBAF). pDule plasmid was generated by amplifying theMjYRS gene from the pBK plasmid isolated from the library using primersRSmovef (5′-CGCGCGCCATGGACGAATTTGAAATG-3′) and RSmover(5′-GACTCAGTCTAGGTACCCGTTTGAAACTGCAGTTATA-3′). The amplified DNAfragments were cloned in to the respective sites on the pDule plasmidsusing the incorporated NcoI and KpnI sites.

Expression and Purification of GFP-1.

DH10B E. coli cells co-transformed with the pBad-sfGFP-134TAG vector andthe machinery plasmid pDule-BIBAF were used to inoculate 5 mL ofnon-inducing medium containing 100 μg/mL Amp and 25 μg/mL Tet. Thenon-inducing medium culture was grown to saturation with shaking at 37°C., and 5.0 mL was used to inoculate 0.5 L autoinduction medium with 100μg/mL Amp, 25 μg/mL Tet, and 1 mM 1 (0.5 L of media grown in 2 L plasticbaffled flasks). After 40 hours of shaking at 37° C., cells werecollected by centrifugation.

The protein was purified using BD-TALON cobalt ion-exchangechromatography. The cell pellet was resuspended in wash buffer (50 mMsodium phosphate, 300 mM sodium chloride, pH 7) containing 1 mg/mLchicken egg white lysozyme, and sonicated 3×1 min while cooled on ice.The lysate was clarified by centrifugation, applied to 6-9 mL bed-volumeresin, and bound for 30 min. Bound resin was washed with >50 volumeswash buffer.

Protein was eluted from the bound resin with 2.5 mL aliquots of elutionbuffer (50 mM sodium phosphate, 300 mM sodium chloride, 150 mM imidazolepH 7) until the resin turned pink and the color of the eluent the columnwas no longer green. The elusions concentrations were check with aBradford protein assay. The protein were desalted into PBS using PD10columns and concentrated with 3000 MWCO centrifuge filters.

The location of incorporation of 1 at site D134 in GFP protein isindicated by the space-filling amino acid (previously Asp) in Scheme 2,FIG. 8. Altering the amino acid at site 134 in a flexible loopunconnected to the chromophore does not affect the stability orfluorescence of GFP.

MS analysis of GFP-1 confirmed the efficient high fidelity incorporationof a single unit of 4-(2′-bromoisobutyramido)phenylalanine into GFP inresponse to an amber stop codon. ESI-MS-T of analysis of sfGFP shows asingle major peak at 27827.0 Da±1 Da while ESI-MS-T of analysis of GFP-1shows a single major peak at 28024.0 Da±1 Da. This shows the expectedmolecular weight difference of 197 Da from native GFP indicating asingle efficient incorporation of 4-(2′-bromoisobutyramido)phenylalanineat the expected site. Each sample did show a small peak at −131±1 Daindicating minor amounts of peptidase-based removal of N-terminalmethionines and +22 sodium adducts. No other peaks were observed thatwould correlate with background incorporation of a natural amino acid.

In summary, the evolved MjRS/tRNACUA pair in pDule-BIBAF allows forsite-specific incorporation of 1 in response to an amber codon. Theimage in Scheme 3, FIG. 9 shows expression levels of GFP-wt frompBad-GFP-His6. Production of GFP-1 from pBad-GFP-134TAG-His6 isdependent on 1 in the growth media: lane 3 without 1 present, lane 4with 1 mM 1 present. The functional protein was purified by Co2+affinity chromatography, separated by SDS-PAGE, and stained withCoomassie.

ATRP Reactions Grafting from GFP-wt and GFP-1.

GFP-wt.

Initiator stock solution: Bpy (16.70 mg, 1.07×10⁻³ mmol) and Cu(II)Br₂(6 mg, 2.68×10⁻⁴ mmol) were dissolved in 10 mL of H₂O; the solution wasdeoxygenated with nitrogen. Cu(I)Br (3.8 mg, 2.68×10⁻⁴ mmol) was addedto the mixture. Monomer, OEO₃₀₀MA (21 mg, 6.9×10⁻² mmol) was added to100 μL of GFP-wt (10.2 mg, 3.4×10⁻² mmol). This solution wasdeoxygenated with nitrogen for 20 min. and then degassed initiatorsolution (250 μl) was added to the reaction mixture. The zero timesample was removed and the reaction was sealed and mixed for 3 hoursthen quenched by exposure to air. There was no difference between GPCtraces from first sample and final sample indicating that no graftingfrom reaction occurred.

GFP-1.

Initiator stock solution: Bpy (16.70 mg, 1.07×10⁻³ mmol) and Cu(II)Br₂(6 mg, 2.68×10⁻⁴ mmol) were dissolved in 10 mL of H₂O; the solution wasdeoxygenated with nitrogen. Cu(I)Br (3.8 mg, 2.68×10⁻⁴ mmol) was addedto the mixture. Monomer, OEO₃₀₀MA (10 mg, 3.42×10⁻² mmol) was added to100 of GFP-1 (6 mg, 2.14×10⁻⁴ mmol). This solution was deoxygenated withnitrogen for 20 min. then degassed initiator solution (100 μL) was addedto the reaction mixture. The reaction was sealed and mixed for 3 hoursthen quenched by exposure to air. The production of GFP-1-p(OEO300MA)was confirmed by FPLC SEC analysis and SDS-PAGE. The reaction appearedto have high initiator efficiency, above 95%, as indicated by the areaunder the curve for the residual GFP-1 in the GFP-p(OEO300MA) sampletaken after 180 min, FIG. 4. As can clearly be seen there is a tailingtowards low Mn region of the elutogram. The primary issue seems to bepoor deactivation since changing the monomer concentration from ˜35% to˜4% in aqueous solutions a tail towards low Mn forms in the low monomerconcentration case.

These protein-polymer hybrids were analyzed using dynamic lightscattering, UV-visible fluorescence spectroscopy and confocal microscopyto confirm the successful incorporation of the protein into the hybridwhile preserving its tertiary structure, FIG. 3.2.

Confirmation of Preparation of a Thermoresponsive Protein-PolymerHybrid.

An interesting phenomenon that is observed with the p(OEO₃₀₀MA) polymersis their LCST behavior at ˜64° C. To determine if the GFP-1-p(OEO₃₀₀MA)hybrids retained this property dynamic light scattering (DLS) wasemployed to study the thermal response of the system. Setting thetemperature to 25° C. and raising the temperature in 1° C. steps adistinct transition from 10 nm (GFP-1-p(OEO₃₀₀MA)) to micron sizedparticles was clearly observed at 64° C. The GFP-1-p(OEO₃₀₀MA) retainedits initial size after cooling to room temperature after 2 iterations ofLEST, FIG. 5. This exemplary thermoresponsive phenomenon of theprotein-polymer conjugate can be made more powerful when hybridstructures with biologically relevant LCST temperature are prepared bycopolymerization procedures for potential controlled releaseapplications.

Example 2: Synthesis of a Protein-g-Polymer Nanogels

In order to exemplify another embodiment, cationic nanogels wereprepared by ATRP in an inverse mini-emulsion in order to improve controlover particle size and prepare functional nanogels between 50 and 200 nmin diameter. Incorporation of a degradable crosslinker allowsbio-degradation of the crosslinkage and release of encapsulatedbiomolecules and colloidal stability. Particle size was measured using aZetasizer Nano from Malvern Instruments. Confocal microscopy was carriedout using a Carl Zeiss LSM 510 Meta NLO Confocor 3 Inverted SpectralConfocal Microscope using an excitation of 488 nm. UV-vis spectroscopywas conducted on a Cary 5000 spectrophotometer and fluorescence spectrawere collected on a Cary Eclipse fluorescence spectrophotometer.

GFP-NG

To prepare the water phase of an inverse miniemulsion ATRP Cu(II)Br₂(2.79 mg, 0.013 mmol) TPMA catalyst (3.63 mg, 0.013 mmol), 4% (w/w totalsolids) GFP-1 (52.5 mg, 0.002 mmol), PEO₂₀₀₀iBBr (50 mg, 0.025 mmol) aco-initiator, oligo(ethylene oxide)₃₀₀methacrylate (OEO₃₀₀MA) (900 mg, 3mmol), as a monomer, and PEO₄₀₀₀dimethacrylate (400 mg, 0.1 mmol) as acrosslinking agent were dissolved in 1.46 ml 0.1M PBS buffer pH 7.4 andemulsified with a 0.05% (w/w) of span-80 in cyclohexane using ultrasonication to form stable droplets ˜200 nm size. After degassing,ascorbic acid was injected to reduce the Cu(II)Br₂ to Cu(I)Br andinitiate polymerization which was stopped after 15 hours stirring at 30°C. The nanogels were purified by precipitation by addition of theemulsion into THF followed by extensive dialysis (50000 MWCO membrane)into water to remove unreacted reagents. Dynamic light scattering ofGFP1-NG peak showed that the particle size ˜240 nm and confocalmicroscopy of GFP1-NG showed that the nanoparticles retained theirfluorescent properties, FIG. 3.2.

Catalase-NG (FIG. 2)

Catalase is an enzyme that converts H₂O₂ into oxygen and water. Acatalase enzyme that was been engineered with 4 ATRP initiating groups,(Cat-1), was employed as one of two different initiators, the other wasa mono-functional PEG based MI, in an ATRP of PEO300MA and a PEG basedcrosslinker, PEG₄₀₀₀DM, to prepare an enzyme-g-PEG conjugatenanoparticle that was evaluated as a reducing agent.

PEO₂₀₀₀-iBBr (56 mg, 0.028 mmol), 238-catalse-initiator (6.3 mg, 0.018μM 0.06 mol %, 4 weight %) OEO₃₀₀MA (1008 mg, 3.36 mmol), PEG₄₀₀₀DM (448mg, 0.11 mmol), CuBr₂ (3.12 mg, 0.014 mmol), TPMA (4.06 mg, 0.014 mmol)were dissolved in 1.46 ml of water in a 50 ml pear shaped flask. A 0.05w/w % solution of Span-80 (1.46 g) in cyclohexane (29.26 g) was added tothe reaction mixture and the solution was sonicated until a stableinverse mini-emulsion was formed. The solution was degassed and 200 μLof degassed ascorbic acid in water (0.066 mg/ml) was added to activatethe catalyst by reducing a fraction of the Cu^(II)/complex to Cu^(I).After 15 hours the solid hybrid was precipitated by addition to THF,washed twice with THF and 3 times with water. The resulting nanogelswere extensive purified using tangential flow filtration with a 300 kDaMWCO membrane.

In both the GFP-1 and Catalase 1 examples well defined protein nanogelswere produced as measured by dynamic light scattering (DLS), FIG. 3.1.Confocal microscopy was used to study the structure of the GFP-nanogeldue to intrinsic light emitting properties of the GFP, FIG. 3.2, todetermine the structural integrity of the protein within the greaternanogel matrix. The retention of the fluorescent properties indicatethat proteins can be subjected to grafting from reaction while retainingtheir shape and biological activity

The Catalase-nanogel could be studied by testing the activity of thisenzyme by exposing it to H₂O₂ to show that it retained catalyticactivity. When hydrogen peroxide was added to an aqueous solution of thenanogels there was an immediate evolution of oxygen, see Image 1, FIG.6.

The primary issue in both of the protein nanogels synthesis was thepossibility that free protein was present in the system. To determine ifany free protein is remaining a through purification was conducted onthese systems followed by a leaching assay to determine how much proteinis released into the supernatant liquid. A protein will be considered tobe covalently incorporated if after extensive washing a constantabsorbance in the nanogels is observed.

For more conclusive proof of protein incorporation into a nanogel theapplication of degradable crosslinkers can be applied. Considering thecase of Cat-NG the free Catalase can easily become trapped into thenanogel while the Cat-1 will be covalently attached. The use ofdegradable crosslinkers during the synthesis of allows stable nanogelsto be synthesized purified and then degraded. After degradation thenanogels synthesized with Cat-1 should contain peaks for theprotein-polymer hybrid while the nanogels synthesized with Cat-wt shouldcontain only peaks for Cat-wt.

What is claimed is:
 1. A protein-polymer composition comprising: a firstprotein with a site-specifically incorporated unnatural amino acidhaving a covalently attached polymer, wherein the unnatural amino acidis represented by formula 2:

wherein R1 and R2 are independently H, C1-C8 alkyl, cycloalkyl,heterocycloalkyl, aryl, or heteroaryl; X is F, Cl, Br, I, N₃,alkoxyamine, or a thiocarbonyl thio moiety; A is O, S, or NR, wherein Ris H, C1-C8 alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl;and n is 0, 1, 2, or 3; or a salt thereof.
 2. The protein-polymercomposition of claim 1, wherein the unnatural amino acid is sitespecifically incorporated into the protein one to five times.
 3. Theprotein-polymer composition of claim 1, wherein the incorporatedunnatural amino acid is an initiator for a controlled radicalpolymerization reaction.
 4. The protein-polymer composition of claim 3,wherein the incorporated unnatural amino acid is an initiator for atomtransfer radical polymerization.
 5. The protein-polymer composition ofclaim 1, wherein the unnatural amino acid is represented by formula 2:

wherein R1 and R2 are independently H, C1-C8 alkyl, cycloalkyl,heterocycloalkyl, aryl, or heteroaryl; X is F, Cl, Br, I, N₃, oralkoxyamine; A is O, S, or NR, wherein R is H, C1-C8 alkyl, cycloalkyl,heterocycloalkyl, aryl, or heteroaryl; and n is 0, 1, 2, or 3; or a saltthereof.
 6. The protein-polymer composition of claim 5, wherein theunnatural amino acid is represented by formula 1:

wherein X is a F, Cl, Br, I, or —N₃, or a salt thereof.
 7. Theprotein-polymer composition of claim 6, wherein X is —N₃, or a saltthereof.
 8. The protein-polymer composition of claim 6, wherein X is Bror Cl, or a salt thereof.
 9. The protein-polymer composition of claim 1,wherein the polymer comprises an incorporated cross-linking moiety. 10.The protein-polymer composition of claim 9, wherein the cross-linkingmoiety comprises a polymerizable group.
 11. The protein-polymercomposition of claim 1, wherein the polymer is degradable.
 12. Theprotein-polymer composition of claim 1, wherein the polymer comprisesrepeating units.
 13. The protein-polymer composition of claim 12,wherein the repeating units are selected from the group comprisingmethacrylates, acrylates, acrylamides, styrenics, andacrylamide-styrenics, or combinations thereof.
 14. The protein-polymercomposition of claim 1, wherein the polymer comprises a copolymer. 15.The protein-polymer composition of claim 14, wherein the copolymercomprises repeating units.
 16. The protein-polymer composition of claim15, wherein the repeating units are selected from the group comprisingmethacrylates, acrylates, acrylamides, styrenics, andacrylamide-styrenics, or combinations thereof.
 17. The protein-polymercomposition of claim 1, comprising a second protein with asite-specifically incorporated unnatural amino acid having a covalentlyattached polymer.
 18. A method for preparing a protein-polymercomposition comprising the steps of: providing a first proteincontaining a site specifically incorporated unnatural amino acidinitiator, wherein the unnatural amino acid is represented by formula 2:

wherein R1 and R2 are independently H, C1-C8 alkyl, cycloalkyl,heterocycloalkyl, aryl, or heteroaryl; X is F, Cl, Br, I, N₃,alkoxyamine, A is O, S, or NR, wherein R is H, C1-C8 alkyl, cycloalkyl,heterocycloalkyl, aryl, or heteroaryl; and n is 0, 1, 2, or 3; or a saltthereof, a polymerization catalyst precursor, and an organic solvent toan aqueous solution to form an emulsion; providing a first radicallypolymerizable monomer to the emulsion; and providing a catalystprecursor reducing agent to the emulsion under conditions suitable toinitiate the controlled radical polymerization.
 19. The method of claim18, wherein the controlled radical polymerization is an atom transferradical polymerization.
 20. The method of claim 18, wherein thepolymerization catalyst precursor is CuX′ and a transition metal ligandspecies, wherein X′ is —Cl₂ or —Br₂.
 21. The method of claim 18, whereinthe reducing agent is ascorbic acid or a salt thereof.
 22. The method ofclaim 18, further comprising the step of: adding a cross-linking agentto the emulsion.
 23. The method of claim 22, wherein the cross-linkingagent comprises methacrylates, acrylates, acrylamides, styrenics, oracrylamide-styrenics, or combinations thereof.
 24. The method of claim18, further comprising the step of: adding a coinitiator to theemulsion.
 25. The method of claim 24, wherein the coinitiator ispolyethyleneglycolisobutyryl bromide.
 26. The method of claim 18,wherein the unnatural amino acid initiator is represented by formula 2:

wherein R1 and R2 are independently H, C1-C8 alkyl, cycloalkyl,heterocycloalkyl, aryl, or heteroaryl; X is F, Cl, Br, I, N₃,alkoxyamine, or a thiocarbonyl thio moiety; A is O, S, or NR, wherein Ris H, C1-C8 alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl;and n is 0, 1, 2, or 3; or a salt thereof.
 27. The method of claim 26,wherein the unnatural amino acid is represented by formula 1:

wherein X is a F, Cl, Br, I, or —N₃, or a salt thereof.
 28. The methodof claim 27, wherein X is —N₃, or a salt thereof.
 29. The method ofclaim 27, wherein X is Br or Cl, or a salt thereof.
 30. The method ofclaim 18, further comprising the step of: adding a copolymer to theemulsion.
 31. The method of claim 18, wherein the radicallypolymerizable monomer is added to the emulsion continuously or in stagesduring the polymerization process.
 32. The method of claim 22, furthercomprising the step of: adding a coinitiator to the emulsion.
 33. Themethod of claim 18, further comprising the step of: adding a secondprotein containing a site specifically incorporated unnatural amino acidinitiator to the emulsion.
 34. A protein-polymer composition comprisinga first protein with a site-specifically incorporated unnatural aminoacid having a covalently attached polymer, the unnatural amino acid isrepresented by formula 1:

wherein X is —N₃, or a salt thereof.
 35. The protein-polymer compositionof claim 34 further comprising a second protein with a site-specificallyincorporated unnatural amino acid having a covalently attached polymer.36. The protein-polymer composition of claim 3, wherein the incorporatedunnatural amino acid is an initiator for a controlled radicalpolymerization reaction and is introduced into the first protein from anorthogonal tRNA/amino acyl-tRNA synthase specific for the unnaturalamino acid initiator.